Figure 5. AK136714 binds HuR and FOXO3. (A) RNA pull-down, silver staining, and mass spectrometry analysis were performed to identify specific proteins that bind with AK136714. (B) RNA pull-down was used to verify that AK136714 bind to HuR. (C, D) RIP experiments verified that AK136714 binds HuR. (E) qPCR was used to detect nuclear AK136714 expression. (F) Bioinformatics analysis was performed to predict the possibility of the interaction between HuR and AK136714. (G) Immunofluorescence was performed to detect the colocalization of AK136714 and HuR. (H) Detection of inflammatory factors (TNF-α, IL-1β and IL-16) by RT-PCR after AK136714 overexpression or knockdown. (I) After inhibiting transcription with actinomycin D, the mRNA expression of each inflammatory factor was detected at different times by qPCR. (J) RNA pull-down was used to verify that AK136714 binds to FOXO3. (K, L) RIP experiments verified that AK136714 binds with FOXO3. (M) Luciferase assay was carried out to detect whether AK136714 silencing or overexpression altered the stimulating effect of FOXO3 on Bim. (N) qPCR was used to assess Bim levels after overexpression or knockdown of AK136714. (n=6, *P < 0.05, **P < 0.01).